Table 1.
Overview of performed experiments.
Fig 1.
Comparison of DNA/RNA yields and RNA quality (inset).
(A) Comparison between the AllPrep standard protocol (APS; described below figure), enzymatic lysis (Enz), enzymatic/bead beating (Bead), and Mirvana extraction protocols (Mirvana). (B) Comparison between different iterations of the AP protocol, examining the effect of preservation method {P}, lysis conditions {L}, and other extraction modification steps {E}. The data presents changes in yield relative to a control, where each column shows the effect of one factor relative to a control that differed in that factor only. The control was extracted using either the AP standard protocol or a modification as indicated by the X-axis labels below the horizontal lines. The factor that was changed is indicated just below the data column. The dotted line indicates no change relative to control. All data represent averages of three extractions, with error bars indicating the 95% confidence interval, except for the effect of lysozyme, which averaged the relative yield effects from multiple samples (from DL, LH, and HR samples, three replicate extractions each). Inset: RIN = RNA integrity number quality (0 (poor)- 10 (high) range) assessment by Bioanalyzer; NT = no preservation treatment.
Fig 2.
Analysis of technical bias to perceived community composition.
(A) NMDS of the 16S rRNA gene amplicon sequencing data based on a Bray-Curtis dissimilarity matrix generated after random subsampling of 2,800 sequences from each sample. Bars indicate the range of coordinates for the three replicate extractions/sequencing datasets per treatment. (B) Phylum level data (top 10 most abundant phyla, fractions of all reads). Number 1–3 between parentheses indicates the sequencing run from which each dataset is derived. APO combines three slightly different treatments that did not result in significantly different taxonomic representations (S2 Fig.). Acronyms: Extraction protocols: APS (standard AllPrep protocol), APO (optimized AllPrep protocol), Enz (Enzymatic protocol), Bead (Bead-beating protocol); Preservation methods: NT (none), RL (RNAlater), RP (RNAprotect), BU (Qiagen Lysis Buffer RLT+); Other modifications: LYS (lysozyme), NQ (No QIAshredder column), TL (bead-beating with TissueLyser). Except for the APO samples, none of the Douglas Lake filters were preserved in RNA protection agents.
Table 2.
Significance analysis of community composition differences.